Blastomycosis is a progressive pulmonary infection of immune-competent and -compromised hosts, but the factors responsible for pathogenicity of the causative fungus Blastomyces dermatitidis are not well understood. We identified BAD1 (formerly WI-1), a surface antigen and adhesin on B. dermatitidis, and developed tools to genetically manipulate B. dermatitidis. On targeting and disrupting BAD1, we found that BAD1 is an essential virulence factor that suppresses host TNF-alpha responses and promotes fungal evasion of immunity. BAD1 displays yeast-phase specific expression, which is regulated transcriptionally by a mechanism shared in B. dermatitidis and Histoplasma capsulatum. Strong preliminary data indicate that BAD1's tandem repeat contributes to virulence, and its C-terminal domain fixes the protein to the yeast surface. We hypothesize that BAD1 is a multi-domain protein whose structural motifs - a core, tandem repeat domain with EF hands for Ca2+ binding and a C terminal EGF-like domain - must be present and assembled properly to mediate pivotal biological functions in the host-pathogen interaction. We also postulate that upstream regulators conserved in B. dermatitidis and H. capsulatum govern expression of BAD1 and other yeast-phase genes. Our 3 specific aims are to: 1) Elucidate the functional role of BAD1 domains, BAD1 deletions and domain swaps will be engineered and expressed in bad1 null B. dermatitidis. These strains and 6 HIS-tagged BAD1 derivatives purified from them, or their synthetic peptides, will be used to define the key domains in pathogenesis, and their modes of action. 2) Define critical features of BAD1 functional domains. Structural bases for virulence and TNF-alpha suppression due to the tandem repeats will be defined. Ca2+ binding by repeats will be studied for effects on BAD1's structure and function. Mutations of the repeat EF hand will assess effects on BAD1 Ca 2+ binding, shape and function. 3) Identify regulators of BAD 1 transcription. Insertional mutants of a reporter strain with a P(BAD1)-lacZ fusion will be screened for impaired P(BAD1) regulation due to insertion into regulatory genes. Regulators of BAD1, other co-regulated phase-specific genes and morphogenesis will be cloned and studied from among the mutated genes. Our work will resolve the molecular and structural bases of Blastomyces virulence and define how BAD1 is regulated and controlled coordinately with other yeast-phase genes, which is a key event in fungal pathogenesis.